Project Summary Pathogenic bacteria need to acquire essential nutrients to establish and sustain an infection in their hosts. The host restricts access to such nutrients, a concept termed nutritional immunity. As such, bacterial nutrient import systems, and the metabolic regulators that govern them, represent next- generation targets for novel antimicrobials. The Gram-positive bacterium B. anthracis is the causative agent of anthrax disease and a weapon of bioterrorism. This pathogen has a remarkable ability to replicate in vertebrates, a virtue that is useful for the study of how bacteria overcome nutritional immunity. It is well known that the penultimate step in the biosynthesis of branched amino acids, which are necessary for all life, requires an enzyme whose activity is dependent on an iron-sulfur cluster. Our published work and preliminary data suggests that B. anthracis and related species employ a clever metabolic mechanism to govern the balance between the intake of branched amino acids and the maintenance of iron homeostasis in complex host environments. Working under the premise that bacillus is auxotrophic for valine when external iron levels are low, and thus cannot make branched amino acids, we hypothesize that host valine liberated from blood proteins stimulates iron acquisition via the global regulator of virulence in Gram-positive bacteria, CodY. This in turn promotes the import of iron, thereby relieving the auxotrophy and fueling rapid replication in host blood and tissues. In Aim 1 of this project, we report the discovery of a novel branched amino acid transporter and characterize its role in the CodY-dependent stimulation of heme-iron acquisition. In Aim 2, we investigate the mechanism of iron release from heme via two newly uncovered heme-binding enzymes in bacillus. Finally, in Aim 3, we integrate key aspects of the central model to determine the importance of these systems in every step of a developing bacillus infection during anthrax disease. Since this network of nutrient uptake systems and regulators also have homologs in several clinically significant species, the work here will provide fundamental knowledge of how pathogenic bacteria fuel their metabolism during virulence.